The invention illustrated herein relates to the combined technique of liquid chromatography (LC)/mass spectrometry (MS) (see for example: Arpino, P. (1992), Mass Spectrom. Rev.,11,3; Blakley, C. R., and Vestal, M. L. (1983), Anal. Chem.,55,750; J. B. Fenn,. M Mann,. C. K. Meng, S. F. Wong, C. M. Whitehouse, Mass Spectrometry Reviews, 1990, 9, 37-70; Liquid Chromatography-Mass Spectrometry: Second Edition, Volume 79, by W. M. A. Niessen, pgs. 135-344 but is also suited for other hyphenated techniques (e.g. gas chromatography/mass spectrometry, liquid chromatography/ultra violet spectroscopy, liquid chromatography/nuclear magnetic resonance spectroscopy). It will also be suitable for other time-resolved spectroscopic techniques, such as direct probe mass spectrometry, laser analysis for spectrometry, fast atom bombardment mass spectrometry. In the case presented for example, the LC is used to separate mixtures into individual components which are in turn passed through to the MS where mass spectral information is obtained on each component. Two or more samples of the same or very similar materials are analyzed under the same conditions. The mass spectral information is used both as a component detection system, and may also be used to characterize the molecular structure of the detected components.
Liquid chromatography itself, is one type of chromatographic technique. Chromatography is a method for separating mixtures. In the simplest application of a chromatographic process, a vertical tube is filled with a finely divided solid known as the stationary phase. The mixture of materials to be separated is placed at the top of the tube and is slowly washed down with a suitable liquid, or eluent, known as the mobile phase.
The mixture first dissolves, each molecule is transported in the flowing liquid, and then becomes attached, or adsorbed, to the stationary solid. Each type of molecule will spend a different amount of time in the liquid phase, depending on its tendency to be adsorbed, so each compound will descend through the tube at a different rate, thus separating from every other compound.
The molecules of the mixture to be separated pass many times between the mobile and stationary phases. The rate at which they do so depends on the mobility of the molecules, the temperature, and the binding forces involved. It is the difference in the time that each type of molecule spends in the mobile phase that leads to a difference in the transport velocity and to the separation of substances. (See FIG. 1a.)
High Pressure Liquid chromatography (HPLC), is a refinement of standard column chromatography. Here, the particles that carry the stationary liquid phase are very small (0.01 mm/0.0004 in) and very uniform in size. For these reasons, the stationary phase offers a large surface area to the sample molecules in the mobile liquid phase. The large pressure drop created in the column filled with such small particles is overcome by using a high-pressure pump to drive the mobile liquid phase through the column in a reasonable time. This method of separation is very reproducible from sample to sample.
Chromatography is used primarily as a separation technique. Despite the reproducible differences in the analysis times for different species noted above, there is generally insufficient specificity to allow identification of the components. For this reason, it is common for chromatographic techniques to be used in tandem with an identification technique, the technique most suitable and most often used being mass spectrometry.
The mass spectrum of a component generally provides a measure of the molecular weight of the component and also provides a characteristic `fingerprint` fragmentation pattern. In a mass spectrometer, the component molecules become ionized and will be excited with a range of energies. Those molecules with least energy generally remain intact and when detected provide a measure of the component's molecular weight. Those molecules ionized with higher amounts of energy will fragment to form smaller product ions characteristic of the molecular structure. To obtain the molecular structure, the fragment ions produced can be pieced together to provide the initial molecular structure. An alternative method for obtaining the molecular structure from the mass spectrum is to compare the spectrum of the component with a large library of reference mass spectra. The unique nature of a component's mass spectrum generally allows ready and unequivocal identification if there is an example of the mass spectrum of that component in the reference library.
For LCMS, the chromatographic device then is interfaced directly to a mass spectrometer which is scanned repetitively (e.g. every 1-5 sec.) as the separated components elute from the chromatograph. In this way a large number of mass spectra are recorded for each analysis. Many of the spectra will record only `background`, i.e. when no components are eluting from the chromatograph. As each component elutes from the chromatograph, the mass spectra will change depending on the nature of the component entering the mass spectrometer. Each mass spectrum produced will contain a certain number of ions, which in turn give rise to an ion current which is plotted against time to produce a total ion chromatogram (TIC)).
An alternative plot is that of an individual mass against time to produce a mass chromatogram which will show just where that particular mass is detected during the analysis. For samples with UV chromophores, an in-line UV detector can be used to detect peaks. Knowing the peak retention times, the corresponding mass spectra can then be obtained. This indirect peak detection method is clearly limited to components with chromophores, which is a serious limitation.
In liquid chromatography/mass spectrometry (LCMS), most of the liquid mobile phase must be removed in the interface region prior to entering the mass spectrometer as mass spectrometers need to operate under high vacuum (See FIG. 1b). However, the liquid mobile phase is present in such excess that the mobile phase is still present in excess to analyte species even after passage through the interface. To obtain good component separations and clean passage of components through a LC column, it is also generally necessary to add buffers to the mobile phase. Hence, mobile phase with associated buffer pass continually through to the mass spectrometer, become ionized and are the major species responsible for the `background` spectra referred to above. Unfortunately, particularly for the popular `spray` LCMS interfacing and ionizing techniques (e.g. electrospray, thermospray), this background varies considerably with time and cannot just be subtracted from analyte spectra. This causes the small to medium level components in the separation to be lost in the high background noise response seen in the TIC.
A flow diagram of a LC-MS experiment is presented (FIG. 2).
The analysis of LC/MS data is a very time consuming and complex process. The intrinsic high background makes it difficult to pick out the lower level components in a mixture. CODA, previously described (U.S. Pat. No. 5,672,869), deals with the chemical noise and provides a high quality, low background data set containing the significant components detected in the sample. Comparison of very similar samples presents further challenges. Even with the use of CODA, it is difficult to see differences between related samples, unless the differences are major components. The problems are illustrated in FIG. 3. The chromatograms of three slightly different samples are shown. Despite the use of CODA, which reduces the number of chromatograms in each file from around 2000 to around 150, the minor differences between the samples are not clear. The separate evaluation of files, which is the only option with commercially available software, is a time consuming task, even after the significant data reduction by CODA.
A new method is needed to extract the differences between two or more closely related samples in order to obtain a faster analysis.